Abstract

Gram-negative bacterial pathogens belonging to the Pasteurellaceae, Moraxellaceae, and Neisseriaceae families rely on an iron acquisition system that acquires iron directly from host transferrin (Tf). The process is mediated by a surface receptor composed of transferrin-binding proteins A and B (TbpA and TbpB). TbpA is an integral outer membrane protein that functions as a gated channel for the passage of iron into the periplasm. TbpB is a surface-exposed lipoprotein that facilitates the iron uptake process. In this study, we demonstrate that the region encompassing amino acids 7-40 of Actinobacillus pleuropneumoniae TbpB is required for forming a complex with TbpA and that the formation of the complex requires the presence of porcine Tf. These results are consistent with a model in which TbpB is responsible for the initial capture of iron-loaded Tf and subsequently interacts with TbpA through the anchor peptide. We propose that TonB binding to TbpA initiates the formation of the TbpB-TbpA complex and transfer of Tf to TbpA.

Kinetics of binding of TbpA and TbpB to immobilized pTf.A–D, the binding curves with increasing concentrations of TbpB (−1) (A), TbpB (−36) (B), His-tagged TbpB (−36) (C), and His-tagged TbpA (D) with pTf immobilized on an SPR biosensor as a function of time are illustrated. Black lines show the experimental data, and the red line shows fitting data. Concentrations of each Tbp are indicated on the respective sensorgrams. The calculated kinetic constants are listed in . r. u, response units.

The intact anchor peptide is required for capture of TbpA by a TbpB-Tf complex. Preparations of His-tagged TbpB from A. pleuropneumoniae containing the intact anchor peptide (left panel) or a truncated anchor peptide (−36TbpB) were mixed with excess non-glycosylated pTf and captured on an Ni-NTA resin, washed to remove excess pTf, and resuspended in detergent-containing buffer. Additional buffer with or without an excess of TbpA (+TbpA) was added to the resin and incubated overnight. The resins were collected by centrifugation and washed twice, and the final pellet was resuspended in SDS-PAGE buffer to elute bound proteins. The samples were applied to an SDS-PAGE gel and stained with Coomassie Blue protein stain.

The intact anchor peptide is required for binding of TbpB to a TbpA-Tf complex. Purified, polyhistidine-tagged, recombinant TbpA from A. pleuropneumoniae strain H49 was mixed with recombinant porcine Tf (left panel) or detergent-containing buffer alone (right panel) and incubated overnight at 4 °C. Buffer without (lanes 1 and 4) or with TbpB with a truncated (37–528) or intact (2–528) N-terminal anchor peptide region was added, and the mixtures were incubated overnight at 4 °C. Ni-NTA resin was added to the incubation mixtures, collected by centrifugation, and washed twice prior to the addition of SDS-PAGE buffer to elute bound proteins.

Defining the region of the anchor peptide required for binding to TbpA. Purified, polyhistidine-tagged, recombinant TbpA from A. pleuropneumoniae strain H49 was mixed with an Ni-NTA resin, and after incubation and washing, recombinant pTf was captured by the immobilized TbpA. The washed resin containing captured TbpA-pTf complex was mixed with different N-terminal truncations of TbpB. After incubation and washing, SDS-PAGE buffer was added to the resin, and the eluted proteins were analyzed by SDS-PAGE and detected by Coomassie Blue stain.

The anchor peptide is not sufficient for binding to TbpA. Preparations of His-tagged anchor peptide (amino acids 2–40) fused to intact TbpB, TbpB N-lobe, or Mbp were captured on an Ni-NTA resin. Holo (iron-loaded) pTf was added to one set of samples (+pTf) prior to the addition of untagged TbpA. After incubation overnight, the resins were collected by centrifugation and washed twice, and the final pellet was resuspended in SDS-PAGE buffer to elute bound proteins. The samples were applied to an SDS-PAGE gel and stained with Coomassie Blue protein stain. MWS, molecular weight standard.

Model for capture of Tf by TbpB. Iron-loaded (holo) Tf is captured by TbpB with the N-terminal anchor region in a fully extended conformation. This places TbpB beyond the lipo oligosaccharide side chains and the polysaccharide capsule. The binding of Tf by the N-terminal lobe of TbpB displaces the anchor peptide from its interaction with the C-terminal lobe and results in conformational changes in the anchor peptide that bring the TbpB-Tf complex closer to the surface of the outer membrane and capable of interacting with “activated” TbpA (bound to TonB). Tf released by TbpB is readily captured by the activated TbpA so that the iron removal and transport process can proceed, resulting in release of apoTf.